The purpose of this study is to compare methylation profiles of LUSC between tumor tissue and normal tissue
Overall design
(1) Genomic DNA quantitation: DNA samples were checked for their quality using NanoDrop® ND-2000 UV-Vis Spectrophotometer. The samples were electrophoresed on agarose gels and those with intact genomic DNA, showing no smear upon agarose gel electrophoresis, were selected for subsequent experiment. Intact genomic DNA was diluted to 50 ng/㎕ based on Quant-iT PicoGreen (Invitrogen) quantitation. Concentrations were adjusted according to these results. All prepared samples were bisulfite-converted according to the EZ DNA methylation kit (Zymo Research) protocols. (2) Bisulfite conversion (Zymo EZ DNA methylation kit): For the bisulfite conversion, 600 ng of input gDNA was required. Conversion reagent was added, followed by subsequent incubation in a thermocycler to denature. CT-converted DNA was washed and de-sulfonated with de-sulfonation buffer, after which, the DNA was washed again and eluted with 12-ml elution buffer. (3) Sample amplification and hybridization for BeadChips: The whole-genome amplification process required 250 ng of input bisulfite-converted DNA, MA1, and created a sufficient quantity of DNA (1000× amplification) to be used on a single BeadChip in the Infinium methylation assay (Illumina RPM and MSM). After amplification, the product was fragmented using a proprietary reagent (FMS), precipitated with 2-propanol (plus precipitating reagent; PM1), and re-suspended in formamide-containing hybridization buffer (RA1). The DNA samples were denatured at 95 °C for 20 min, and placed in a humidified container for a minimum of 16 h at 48 °C, allowing CpG loci to hybridize with the 50-mer capture probes. (4) Allele-specific single-base extension and staining on BeadChips: Following hybridization, the BeadChip/Te-Flow chamber assembly was placed on a temperature-controlled Tecan flow-through chamber rack, and subsequent washing, extension, and staining were performed by the addition of reagents to the Te-Flow chamber. For the allele-specific single-base extension assay, primers were extended with a polymerase and labeled nucleotide mix (TEM), and stained with repeated application of STM (staining reagent) and ATM (anti-staining reagent). After completion of staining, the slides were washed with low-salt wash buffer (PB1), immediately coated with XC4, and then imaged in the iScan System. (5) Imaging the BeadChip and data analysis: The iScan System has a two-color (532 nm/658 nm) confocal fluorescent scanner with 0.54-μm pixel resolution. The scanner excited the fluorophores generated during signal amplification/ staining of the allele-specific (one color) extension products on the BeadChips. The image intensities were extracted using Illumina’s GenomeStudio Software.
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